In the biological production of commercially viable proteins by the fermentation of microorganisms, the ability to produce the desired proteins by fermentation with secretion of the proteins by the microorganisms into the broth is very significant. However, there are many commercially viable proteins encoded by genetically engineered DNA constructs which are not secreted by the cells in which the DNA is expressed. This often necessitates harvesting the cells, bursting the cell walls, recovering the desired proteins in pure form and then chemically re-naturing the pure material to restore its bioactive function. This downstream processing, as it is called, is illustrated in FIG. 1.
Some cells and microorganisms carry out the biological equivalent of downstream processing by secreting proteins in bioactive form. The mechanism which directs the secretion of some proteins through the cell walls is not fully understood. For example, in Streptomyces griseus, an organism used for the commercial production of Pronase, the species secretes many extra cellular proteins (Jurasek, L., P. Johnson, R. W. Olafson, and L. B. Smillie (1971), An improved fractionation system for pronase on CM-Sephadex Can. J. Biochem., 49:1195-1201). Protease A and protease B, two of the serine proteases secreted by S. griseus, have sequences which are 61% homologous on the basis of amino acid identity (Fujinaga. M., L. T. J. Delbaere, G. D. Brayer, and M. N. G. James (1985), Refined structure of .alpha.-lytic protease at 1.7A resolution; Analysis of hyrodgen bonding and solvent structure, J. Mol. Biol., 183:479-502; Jurasek, L., M. R. Carpenter, L. B. Smillie, A. Gertler, S. Levy, and L. H. Ericsson (1974), Amino acid sequencing of Streptomyces griseus protease B, A major component of pronase, Biochem. Biophys. Res. Comm., 61:1095-1100; Young, C. L., W. C. Barker, C. M. Tomaselli, and M. O. Dayhoff (1978), Serine proteases, In M. O. Dayhoff (ed.), Atlas of Protein Sequence and Structure 5, suppl. 3:73-93). These proteases also have similar tertiary structure, as determined by X-ray crystallography (Delbaere, L. T. J., W. L. B. Hutcheon, M. N. G. James, and W. E. Thiessen (1975), Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes, Nature 257:758-763; Fujinaga. M., L. T. J. Delbaere, G. D. Brayer, and M. N. G. James (1985), Refined structure of .alpha.-lytic protease at 1.7A resolution; Analysis of hyrodgen bonding and solvent structure, J. Mol. Biol., 183:479-502; James, M. N. G., A. R. Sielecki, G. D. Brayer, L. T. J. Delbaere, and C.-A. Bauer (1980), Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8. A resolution, J. Mol. Biol., 144:43-88). Although the structures of proteases A and B have been extensively studied, the genes encoding these proteases have not been characterized before.